Single‐cell profiling reveals novel cellular heterogeneity of monocytes during Hymenoptera venom allergy

Abstract Background Hymenoptera stings can induce dysregulated inflammation and immediate hypersensitivity reactions including anaphylaxis. However, the molecular mechanisms underlying peripheral immune responses during Hymenoptera venom allergy (HVA) remain elusive. Methods Here we determined the single‐cell transcriptomic profiling from highly heterogeneous peripheral blood cells in patients with HVA through unbiased single‐cell RNA sequencing and multiple models of computational analyses. Results Through clustering analysis by uniform manifold approximation and projection, we revealed an increased number of monocytes in the acute phase and identified innate immune responses, leukocyte activation, and cellular detoxification as the main involved biological processes. We used filter analysis to identify that CLU that encodes clusterin was highly expressed in monocytes, and the co‐expressed genes of CLU further supported the key role of monocyte. We further used pseudo‐temporal ordering of cells and scRNA velocity analysis to delineate disease‐associated monocyte lineages and states in patients with HVA. Conclusions Our comprehensive molecular profiling of blood samples from patients with HVA revealed previously unknown molecular changes, providing important insights into the mechanism of venom allergy and potential therapeutic targets.


| TO THE EDITOR
The immune response induced by Hymenoptera stings can cause dysregulated inflammation and systemic allergic responses. 1 A wide range of clinical manifestations including anaphylactic shock result from Hymenoptera venom allergy (HVA). 2 In Europe, prevalence of systemic anaphylactic sting reactions ranges from 0.3% to 7.5%. 3 The current emergency treatment can decelerate the development of systemic allergic reactions by injecting non-specific immune regulators, such as corticosteroids. 4 However, even if the pathogenic mechanism is inferred from previous observations, molecular mechanisms underlying peripheral immune responses during HVA are elusive, which is mainly because few genomics datasets are available for understanding the immune response caused by Vespa stings.
Here we determined the blood single-cell signatures from highly heterogeneous peripheral blood cells in patients with HVA through unbiased single-cell RNA sequencing and multiple models of computational analyses.
To investigate how the molecular and cellular profiles of blood leukocytes are altered in patients with HVA compared to those in healthy control (HC) individuals, we performed single-cell transcriptome analysis of 11 blood leukocyte samples (n = 7892 cells) by single-cell RNA sequencing (scRNA-seq). Four HVA blood samples (n = 2056 cells) were obtained from two HVA patients (P1, n = 1503 cells; P2, n = 553 cells), one sample each at acute and recovery stages. Seven HC blood samples (n = 5836 cells) were obtained from seven HC individuals.
To establish a baseline profile of the cell populations, we performed an initial unbiased uniform manifold approximation and projection (UMAP) clustering using all 11 samples ( Figure 1A). This analysis generated four major cell clusters that were subsequently , we identified S100A8, S100A9, and S100A12 genes as co-expressed genes of CLU gene in HVA patients, which also confirmed the roles of the selected DEGs based on the pathway analysis. These gene expression network may also support the roles of clusterin involved in the strong inflammatory processes 7 or various stresses such as oxidative stress 8 in human diseases.
To determine whether hypothetical developmental relationships exist between PBMCs, we performed single-cell trajectory analysis to identify the pseudo-temporal ordering of cells based on single-cell transcriptomic datasets. A total of 13 marker genes (four HVAassociated genes and nine CLU-co-expressed genes) were found to be involved in pseudo-temporal ordering of cells into three different branches according to their characteristics. This pseudo-temporal ordering was found to be applicable to defining the root nodes and direction of the trajectory ( Figure S1). Notably, using pseudo-temporal ordering analysis, CLU + cells were classified into the same group as CES1 + cells and PLAC8 + cells, suggesting that the properties of disease-associated cells can be identified from our feature selection strategies. To further investigate the dynamic changes in cell transitions and differentiation, we applied scVelo (a tool for analyzing RNA velocity analysis at the single-cell level) 9 to determine the direction of the monocyte cell population transition by evaluating the abundance of unspliced (nascent) and spliced (mature) mRNA. Analysis of RNA velocity information within the subset of three monocyte subsets (M1, M2, and M3 clusters) from all PBMCs in HVA patients indicated differentiation of M1 or M2 clusters toward the M3 cluster ( Figure S2a).
Moreover, analysis of RNA velocity information for three stages (acute, recovery, and HC) from all monocytes indicated the unique differentiation status of monocytes during the acute stage ( Figure S2b).
These findings supported that this differentiation of monocytes can be affected by stimulation with Vespa stings.

| CONCLUSION
Our comprehensive molecular profiling of blood samples from patients with HVA revealed previously unknown molecular changes, providing important insights into mechanisms of venom allergy and potential therapeutic targets for precision medicine in HVA.

KEYWORDS
immunology, immunotherapy, insect hypersensitivity, molecular allergy Wen-Cheng Chao and Wen-Ting Liao contributed equally to this work.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.